AMP-activated protein kinase (AMPK) plays a major role in the response of many cells to stresses that alter their energy state. In skeletal muscle, AMPK activity is increased during exercise (contraction), and a large body of evidence indicates that this regulates observed increases in glucose transport, fatty acid oxidation and UCP3 expression. Much of this knowledge has been obtained utilizing AICAR, an AMPK activator whose specificity has been questioned. Here, we will make use of AICAR and alternative approaches to modulate AMPK activity, to explore the physiological roles of AMPK in muscle and the biochemical basis for its effects on glucose transport, glycogen synthesis and UCP3 expression. The following are our aims: (1) To characterize pharmacological and other means for increasing AMPK activity in incubated muscles and to determine if they stimulate glucose transport via nitric oxide. The effects of prior hypoxia, incubation with a glucose-free medium, metformin and alpha-adrenergic stimulation, all of which increase AMPK activity, will be compared with that of AICAR. In addition, we will assess the specificity of a new small molecular weight AMPK inhibitor. (2) To determine the biochemical pathway(s) by which AMPK activation in skeletal muscle leads to GLUT4 translocation and UCP-3 gene transcription. In vitro phosphorylation assays and metabolic labeling of isolated muscle will be used to identify the phosphoprotein targets of AMPK related to glucose transporter translocation. We will also introduce constitutively active and dominant negative AMPK constructs into cultured myocytes to ascertain the AMPK-dependency of the UCP3 mRNA induction, and we will attempt to identify the transcription factors that modify this process. (3) To explore the basis for the inhibition of insulin-stimulated glycogen synthesis in the denervated extensor digitorum longus (EDL) muscle and its restoration by incubation with AICAR. We will determine whether the effects of AICAR are mimicked by other AMPK activators, what processes AMPK activation restores and why AICAR does not have a similar effect in the denervated soleus. (4) To determine whether treatment with AICAR or metformin in vivo prevents or attenuates the alterations in gene expression (e.g. GLUT4, UCP-3, myogenin, etc.), and lipids that occur in rat muscle 6-72 hrs after denervation. We will also assess if treatment with these agents stimulates GLUT4 recruitment, and we will use new methodology to test the hypothesis that exercise/contraction/AMPK responsive glucose transporter pools differ from insulin-responsive pools in their biochemical composition and ability to associate with glycogen particles.